Plant amino acid biosynthetic enzymes

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a plant cysteine γ synthase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the plant cysteine γ synthase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the plant biosynthetic enzyme in a transformed host cell.

This application is a continuation-in-part of application Ser. No.09/424,976 filed on Dec. 2, 1999 which is a national stage applicationof PCT/US98/12073 with an International filing date of Jun. 11, 1998,which in turn claims priority benefit of U.S. Provisional ApplicationNo. 60/049,406, filed Jun. 12, 1997 and U.S. Provisional Application No.60/065,385, filed Nov. 12, 1997.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingenzymes involved in amino acid biosynthesis in plants and seeds.

BACKGROUND OF THE INVENTION

Many vertebrates, including humans, lack the ability to manufacture anumber of amino acids and therefore require these amino acids in theirdiet. These are called essential amino acids. Grain-derived foods orfeed, however, are deficient in certain essential amino acids, such aslysine, the sulfur-containing amino acids methionine and cysteine,threonine and tryptophan. For example, in corn (Zea mays L.) lysine isthe most limiting amino acid for the dietary requirements of manyanimals, and soybean (Glycine max L.) meal is used as an additive tocorn-based animal feeds primarily as a lysine supplement. Oftenmicrobial-fermentation produced lysine is needed for suchsupplementation. Thus, an increase in lysine content of either corn orsoybean would reduce or eliminate the need to supplement mixed grainfeeds with lysine produced via fermentation.

Furthermore, in corn the sulfur amino acids are the third most limitingamino acids, after lysine and tryptophan, for the dietary requirementsof many animals. Legume plants, however, while rich in lysine andtryptophan, have low sulfur-containing amino acid content. Therefore,the use of soybean meal to supplement corn in animal feed is notsatisfactory. An increase in the sulfur amino acid content of eithercorn or soybean would improve the nutritional quality of the mixturesand reduce the need for further supplementation through addition of moreexpensive methionine.

One approach to increasing the nutritional quality of human foods andanimal feed is to increase the production and accumulation of specificfree amino acids via genetic engineering of the biosynthetic pathway ofthe essential amino acids. Biosynthetically, lysine, threonine,methionine, cysteine and isoleucine are all derived from aspartate.Regulation of the biosynthesis of each member of this family isinterconnected (see FIG. 1). The organization of the pathway leading tobiosynthesis of lysine, threonine, methionine, cysteine and isoleucineindicates that over-expression or reduction of expression of genesencoding, inter alia, aspartic semialdehyde dehydrogenase, homoserinekinase, diaminopimelate decarboxylase, cysteine synthase andcystathionine β-lyase in corn and soybean could be used to alter levelsof these amino acids in human food and animal feed. However, few of thegenes encoding enzymes that regulate this pathway in plants, especiallycorn and soybeans, are available. Accordingly, availability of nucleicacid sequences encoding all or a portion of these enzymes wouldfacilitate development of nutritionally improved crop plants.

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 42, 44, 46, 48, 50, 8, 10,12, 14, 16, 18, 53, 55, 21, 23, 25, 27, 58, 30, 61, 63, 33, 35, 37, 39,67, 69, and 71.

The present invention concerns isolated polynucleotides comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding a polypeptide of at least 60 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:2, 4, 6, 43, 45, 47, 49, and 51; (b) a nucleotide sequenceencoding a polypeptide of at least 60 amino acids having at least 95%identity based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of SEQ ID NOs:9, 11, 13,15, 17, 19, 54 and 56; (c) a nucleotide sequence encoding a polypeptideof at least 60 amino acids having at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs:22, 24, 26, 28, and 59; (d) anucleotide sequence encoding a polypeptide of at least 60 amino acidshaving at least 95% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:31, 62, and 64; and (e) a nucleotide sequence encoding apolypeptide of at least 60 amino acids having at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:34, 36, 38, 40, 68, 70,and 72. It is preferred that the identity be at least 85%, morepreferably at least 90%, still more preferably at least 95%. Thisinvention also relates to the isolated complement of suchpolynucleotides, wherein the complement and the polynucleotide consistof the same number of nucleotides, and the nucleotide sequences of thecomplement and the polynucleotide have 100% complementarity.

In a third embodiment nucleotide sequence of the isolated firstpolynucleotide is selected from SEQ ID NOs:1, 3, 5, 42, 44, 46, 48, 50,SEQ ID NOs:8, 10, 12, 14, 16, 18, 53 and 55, SEQ ID NOs:21, 23, 25, 27,and 58, SEQ ID NOs:30, 61, and 63, and SEQ ID NOs:33, 35, 37, 39, 67,69, and 71.

In a fourth embodiment, this invention concerns an isolatedpolynucleotide encoding an aspartic semialdehyde dehydrogenase, adiaminopimelate decarboxylase, a homoserine kinase, a cysteine γsynthase or a cystathionine β-lyase.

In a fifth embodiment, this invention relates to a chimeric genecomprising the polynucleotide of the present invention.

In a sixth embodiment, the present invention concerns an isolatednucleic acid molecule that comprises at least 180 nucleotides andremains hybridized with the isolated polynucleotide of the presentinvention under a wash condition of 0.1×SSC, 0.1% SDS, and 65° C.

In a seventh embodiment, the invention also relates to a host cellcomprising a chimeric gene of the present invention or an isolatedpolynucleotide of the present invention. The host cell may beeukaryotic, such as a yeast cell or a plant cell, or prokaryotic, suchas a bacterial cell. The present invention may also relate to a viruscomprising an isolated polynucleotide of the present invention or achimeric gene of the present invention.

In an eighth embodiment, the invention concerns a transgenic plantcomprising a polynucleotide of the present invention.

In a ninth embodiment, the invention relates to a method fortransforming a cell by introducing into such cell the polynucleotide ofthe present invention, or a method of producing a transgenic plant bytransforming a plant cell with the polynucleotide of the presentinvention and regenerating a plant from the transformed plant cell.

In a tenth embodiment, the invention concerns a method for producing anucleotide fragment by selecting a nucleotide sequence comprised by apolynucleotide of the present invention and synthesizing apolynucleotide fragment containing the nucleotide sequence. It isunderstood that the nucleotide fragment may be produced in vitro or invivo.

In an eleventh embodiment the invention concerns an isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) a polypeptide of at least 60 amino acids and having a sequenceidentity of at least 80% based on the Clustal method of alignment whencompared to an amino acid sequence selected from the group consisting ofSEQ ID NOs:2, 4, 6, 43, 45, 47, 49, and 51; (b) a polypeptide of atleast 60 amino acids having a sequence identity of at least 95% based onthe Clustal method of alignment when compared to an amino acid sequenceselected from the group consisting of SEQ ID NOs:9, 11, 13, 15, 17, 19,54 and 56; (c) a polypeptide of at least 60 amino acids having asequence identity of at least 80% based on the Clustal method ofalignment when compared to an amino acid sequence selected from thegroup consisting of SEQ ID NOs:22, 24, 26, 28, and 59; (d) polypeptideof at least 60 amino acids having an identity of at least 95% based onthe Clustal method of alignment when compared to an amino acid sequenceselected from the group consisting of SEQ ID NOs:31, 62, and 64; and (e)a polypeptide of at least 60 amino acids having a sequence identity ofat least 85% based on the Clustal method of alignment when compared toan amino acid sequence selected from the group consisting of SEQ IDNOs:34, 36, 38, 40, 68, 70, and 72. It is preferred that the identity beat least 85%, it is more preferred if the identity is at least 90%, itis preferable that the identity be at least 95%.

In a twelfth embodiment the invention relates to an isolatedpolypleptide selected from SEQ ID NOs:2, 4, 6, 43, 45, 47, 49, and 51,SEQ ID NOs:9, 11, 13, 15, 17, 19, 54 and 56, SEQ ID NOs:22, 24, 26, 28,and 59, SEQ ID NOs:31, 62, and 64, and SEQ ID NOs:34, 36, 38, 40, 68,70, and 72.

In a thirteenth embodiment, this invention concerns an isolatedpolypeptide having aspartic semialdehyde dehydrogenase, diaminopimelatedecarboxylase, homoserine kinase, cysteine γ synthase, or cystathionineβ-lyase function.

In a fourteenth embodiment, this invention relates to a method ofaltering the level of expression of a plant biosynthetic enzyme in ahost cell comprising: transforming a host cell with a chimeric gene ofthe present invention; and growing the transformed host cell underconditions that are suitable for expression of the chimeric gene.

A further embodiment of the instant invention is a method for evaluatinga compound for its ability to inhibit the activity of a plantbiosynthetic enzyme selected from the group consisting of asparticsemialdehyde dehydrogenase, diaminopimelate decarboxylase, homoserinekinase, cysteine γ synthase and cystathionine β-lyase, the methodcomprising the steps of: (a) transforming a host cell with a chimericgene comprising a nucleic acid fragment encoding a plant biosyntheticenzyme selected from the group consisting of aspartic semialdehydedehydrogenase, diaminopimelate decarboxylase, homoserine kinase,cysteine synthase and cystathionine β-lyase, operably linked toregulatory sequences; (b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of thebiosynthetic enzyme in the transformed host cell; (c) optionallypurifying the biosynthetic enzyme expressed by the transformed hostcell; (d) treating the biosynthetic enzyme with a compound to be tested;and (e) comparing the activity of the biosynthetic enzyme that has beentreated with a test compound to the activity of an untreatedbiosynthetic enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 depicts the biosynthetic pathway for the aspartate family ofamino acids. The following abbreviations are used: AK=aspartokinase;ASADH=aspartic semialdehyde dehydrogenase; DHDPS=dihydrodipicolinatesynthase; DHDPR=dihydrodipicolinate reductase; DAPEP=diaminopimelateepimerase; DAPDC=diaminopimelate decarboxylase; HDH=homoserinedehydrogenase; HK=homoserine kinase; TS=threonine synthase; TD=threoninedeaminase; CγS=cystathionine γ-synthase; CβL=cystathionine β-lyase;MS=methionine synthase; CS=cysteine synthase; andSAMS=S-adenosylmethionine synthase.

FIGS. 2 through 6 show the amino acid sequence alignments between theknown art sequences for aspartic semialdehyde dehydrogenase,diaminopimelate decarboxylase, homoserine kinase, cysteine γ synthase,and cystathione β-lyase with the sequences included in this application.Alignments were performed using the Clustal a logarithm described inHiggins and Sharp (1989) (CABIOS 5:151-153). Amino acids conserved amongall sequences are indicated by an asterisk (*) above the alignment.Dashes are used by the program to maximize the alignment. A descriptionof FIGS. 2 through 6 follows:

FIG. 2 shows a comparison of the aspartic semialdehyde dehydrogenaseamino acid sequences from corn contig assembled from clonesp0003.cgpha22r:fis, cpe1c.pk009.b24, p0016.ctscp83r, and p00075.cslab16r(SEQ ID NO:43), rice clone rlr48.pk0003.d12 (SEQ ID NO:2), the contig of5′ RACE PCR and rice clone rlr48.pk0003.d12 (SEQ ID NO:45), soybeanclones sfl1.pk0122.f9 (SEQ ID NO:6), ses9c.pk001.a15:fis (SEQ ID NO:47),and sfl1.pk0122.f9:fis (SEQ ID NO:49), wheat clones wr1.pk0004.cl 1 (SEQID NO:4) and wdk1c.pk014.n5:fis (SEQ ID NO:51) with the Legionellapneumophila (NCBI General Identifier No. 2645882; SEQ ID NO:7) and theAquifex aeolicus sequences (NCBI General Identifier No. 6225258; SEQ IDNO:52). FIG. 2A: positions 1 through 120; FIG. 2B: positions 121 through240; FIG. 2C: positions 241 through 360; FIG. 2D: positions 361 through392.

FIG. 3 shows a comparison of the diaminopimelate decarboxylase aminoacid sequences derived from corn clones cen3n.pk0067.a3 (SEQ ID NO:9)and cr1n.pk0103.d8 (SEQ ID NO:11), rice clone rl0n.pk0013.b9 (SEQ IDNO:13), soybean clones sr1.pk0132.cl (SEQ ID NO:15), sdp3c.pk001.o15(SEQ ID NO:19) and sdp3c.pk001.o15:fis (SEQ ID NO:54), wheat cloneswlk1.pk0012.c2 (SEQ ID NO:17) and wlk1.pk0012.c2:fis (SEQ ID NO:56) withthe Pseudomonas aeruginosa (NCBI General Identifier No. 118304; SEQ IDNO:20) and Arabidopsis thaliana sequences (NCBI General Identifier No.9279586; SEQ ID NO:57). FIG. 3A: positions 1 through 120; FIG. 3B:positions 121 through 240; FIG. 3C: positions 241 through 360; FIG. 3D:positions 361 through 480; FIG. 3E: positions 481 through 535.

FIG. 4 shows a comparison of the homoserine kinase amino acid sequencesderived from corn clone cr1n.pk0009.g4 (SEQ ID NO:22), rice clonesrca1c.pk005.k3 (SEQ ID NO:24) and rca1c.pk005.k3:fis (SEQ ID NO:59),soybean clone ses8w.pk0020.b5 (SEQ ID NO:26), wheat clone wl1n.pk0065.f2(SEQ ID NO:28) with the Methanococcus jannaschii (NCBI GeneralIdentifier No. 1591748; SEQ ID NO:29) and the Arabidopsis thalianasequences (NCBI General Identifier No. 4927412; SEQ ID NO:60). FIG. 4A:positions 1 through 180; FIG. 4B: positions 181 though 360; FIG. 4C:positions 361 through 396.

FIG. 5 shows a comparison of the cysteine γ synthase amino acidsequences derived from the corn contig assembled from clones cco1n.pk083j4, chp2.pk0016.b1, cpd1c.pk004.b20, cr1n.pk0083.c5, csi1.pk0003.g6, andp0126.cn1cb49r (SEQ ID NO:62), rice clone rls6.pk0068.b7:fis (SEQ IDNO:64), soybean clone se3.05h06 (SEQ ID NO:31) with the Citrulluslanatus sequence (NCBI General Identifier No. 540497; SEQ ID NO:32), theSpinacia oleracea sequence (NCBI General Identifier No. 540497; SEQ IDNO:65), and the Solanum tuberosum sequence (NCBI General Identifier No.11131628; SEQ ID NO:66). FIG. 5A: positions 1 through 180; FIG. 5B:positions 181 through 360; FIG. 5C: positions 361 through 424.

FIG. 6 shows a comparison of the amino acid sequences of thecystathionine β-lyase derived from corn clone cen1.pk0061.d4 (SEQ IDNO:34), corn contig assembled from clones p0005.cbmei71r,p0014.ctuui39r, p0109.cdadg47r, and p0125.czaay16r (SEQ ID NO:68), riceclone rlr12.pk0026.g1 (SEQ ID NO:36), the contig of 5′ PCR and riceclone rlr12.pk0026.g1:fis (SEQ ID NO:70), soybean clone sfl1.pk0012.c4(SEQ ID NO:38), and wheat clones wr1.pk0091.g6 (SEQ ID NO:40) andwr1.pk0091.g6:fis (SEQ ID NO:72) with the Arabidopsis thaliana sequence(NCBI General Identifier No. 1708993; SEQ ID NO:41). FIG. 6A: positions1 through 120; FIG. 6B: positions 121 through 240; FIG. 6C: positions241 through 360; FIG. 6D: positions 361 through 483.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Plant Biosynthetic Enzymes SEQ ID NO: (Amino Polypeptide Clone(Nucleotide) Acid) rice ASADH rlr48.pk0003.d12 1 2 wheat ASADHwr1.pk0004.c11 3 4 soybean ASADH sfl1.pk0122.f9 5 6 L. pneumophila ASADHNCBI GI 2645882 7 corn DAPEP cen3n.pk0067.a3 8 9 corn DAPEPcr1n.pk0103.d8 10 11 rice DAPEP rl0n.pk0013.b9 12 13 soybean DAPEPsr1.pk0132.c1 14 15 wheat DAPEP wlk1.pk0012.c2 16 17 soybean DAPEPsdp3c.pk001.o15 18 19 P. aeruginosa DAPEP NCBI GI 118304 20 corn HKcr1n.pk0009.g4 21 22 rice HK rca1c.pk005.k3 23 24 soybean HKses8w.pk0020.b5 25 26 wheat HK wl1n.pk0065.f2 27 28 M. jannaschii HKNCBI GI 1591748 29 soybean CγS se3.05h06 30 31 C. lanatus CγS NCBI GI540497 32 corn CβL cen1.pk0061.d4 33 34 rice CβL rlr12.pk0026.g1 35 36soybean CβL sfl1.pk0012.c4 37 38 wheat CβL wr1.pk0091.g6 39 40 A.thaliana CβL NCBI GI 1708993 41 corn ASADH Contig of: 42 43p0003.cgpha22r:fis cpe1c.pk009.b24 p0016.ctscp83r p0075.cslab16r riceASADH 5′ RACE PCR + 44 45 rlr48.pk0003.d12 soybean ASADHses9c.pk001.a15:fis 46 47 soybean ASADH sfl1.pk0122.f9:fis 48 49 wheatASADH wdk1c.pk014.n5:fis 50 51 A. aeolicus ASADH NCBI GI 6225258 52soybean DAPEP sdp3c.pk001.o15:fis 53 54 wheat DAPEP wlk1.pk0012.c2:fis55 56 A. thaliana DAPEP NCBI GI 9279586 57 rice HK rca1c.pk005.k3:fis 5859 A. thaliana HK NCBI GI 4927412 60 corn CγS Contig of: 61 62cco1n.pk083.j4 chp2.pk0016.b1 cpd1c.pk004.b20 cr1n.pk0083.c5csi1.pk0003.g6 p0126.cnlcb49r rice CγS rls6.pk0068.b7:fis 63 64 S.oleracea CγS NCBI GI 416869 65 S. tuberosum CγS NCBI GI 11131628 66 cornCβL Contig of: 67 68 p0005.cbmei71r p0014.ctuui39r p0109.cdadg47rp0125.czaay16r rice CβL 5′RACE PCR + 69 70 rlr12.pk0026.g1:fis wheat CβLwr1.pk0091.g6:fis 71 72

The nucleotide and amino acid sequences shown in SEQ ID NOs:1 through 41are found, with the same SEQ ID NO, in U.S. application Ser. No.09/424,976. All or a portion of some of the sequences in the presentapplication are found in the provisional applications for which thepresent application claims priority to. Table 1A indicates the SEQ IDNO: in the present application and the corresponding SEQ ID NO: in thepreviously-filed provisional application.

TABLE 1A Sequence Priority Application Provisional ApplicationProvisional Application No. 09/424,976 No. 60/049406 No. 60/065385 SEQID NO: 1 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO:3* SEQ ID NO: 4 SEQ ID NO: 4* SEQ ID NO: 8 SEQ ID NO: 7 SEQ ID NO: 8 SEQID NO: 9 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 12 SEQ ID NO: 9 SEQ ID NO:13 SEQ ID NO: 10 SEQ ID NO: 14 SEQ ID NO: 11 SEQ ID NO: 5 SEQ ID NO: 15SEQ ID NO: 12 SEQ ID NO: 6 SEQ ID NO: 21 SEQ ID NO: 13 SEQ ID NO: 10*SEQ ID NO: 22 SEQ ID NO: 14 SEQ ID NOs: 11* and 14* SEQ ID NO: 23 SEQ IDNO: 17* SEQ ID NO: 15 SEQ ID NO: 24 SEQ ID NO: 18* SEQ ID NO: 16 SEQ IDNO: 25 SEQ ID NO: 15 SEQ ID NO: 13 SEQ ID NO: 26 SEQ ID NO: 16 SEQ IDNO: 14 SEQ ID NO: 30 SEQ ID NO: 19 SEQ ID NO: 17 SEQ ID NO: 31 SEQ IDNO: 20 SEQ ID NO: 18 SEQ ID NO: 33* SEQ ID NO: 21 SEQ ID NO: 19 SEQ IDNO: 34 SEQ ID NO: 22 SEQ ID NO: 20 SEQ ID NO: 37 SEQ ID NO: 23 SEQ IDNO: 21* SEQ ID NO: 38 SEQ ID NO: 24 SEQ ID NO: 22* *Indicates that onlya portion of the sequence was in the application.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acidsequence,” and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 30contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 60 contiguous nucleotides derived from SEQ IDNOs:1, 3, 5, 42, 44, 46, 48, 50, SEQ ID NOs:8, 10, 12, 14, 16, 18, 53and 55, SEQ ID NOs:21, 23, 25, 27, and 58, SEQ ID NOs:30, 61, and 63,and SEQ ID NOs:33, 35, 37, 39, 67, 69, and 71, or the complement of suchsequences.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences with which it isnormally associated such as other chromosomal and extrachromosomal DNAand RNA. Isolated polynucleotides may be purified from a host cell inwhich they naturally occur. Conventional nucleic acid purificationmethods known to skilled artisans may be used to obtain isolatedpolynucleotides. The term also embraces recombinant polynucleotides andchemically synthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides derived from the instantnucleic acid fragment can be constructed and introduced into a plant orplant cell. The level of the polypeptide encoded by the unmodifiednucleic acid fragment present in a plant or plant cell exposed to thesubstantially similar nucleic fragment can then be compared to the levelof the polypeptide in a plant or plant cell that is not exposed to thesubstantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby using nucleic acid fragments that do not share 100% sequence identitywith the gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not affect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 30 (preferably at least 40, mostpreferably at least 60) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 42,44, 46, 48, 50, SEQ ID NOs:8, 10, 12, 14, 16, 18, 53 and 55, SEQ IDNOs:21, 23, 25, 27, and 58, SEQ ID NOs:30, 61, and 63, and SEQ IDNOs:33, 35, 37, 39, 67, 69, and 71 and the complement of such nucleotidesequences may be used in methods of selecting an isolated polynucleotidethat affects the expression of an aspartic-semialdehyde dehydrogenase, adiaminopimelate decarboxylase, a homoserine kinase, a cysteine γsynthase, or a cystathionine β-lyase polypeptide in a host cell. Amethod of selecting an isolated polynucleotide that affects the level ofexpression of a polypeptide in a host cell may comprise the steps of:constructing an isolated polynucleotide of the present invention or anisolated chimeric gene of the present invention; introducing theisolated polynucleotide or the isolated chimeric gene into a host cell;measuring the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of a polypeptide or enzyme activity in ahost cell that does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1 X SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually, by one skilled in the art,or by using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a nucleic acid fragment to beexpressed in most cell types at most times are commonly referred to as“constitutive promoters”. New promoters of various types useful in plantcells are constantly being discovered; numerous examples may be found inthe compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15: 1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“Translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

“3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single polynucleotide so that the functionof one is affected by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Mature protein” or the term “mature” when used in describing a proteinrefers to a post-translationally processed polypeptide; i.e., one fromwhich any pre- or propeptides present in the primary translation producthave been removed. “Precursor protein” or the term “precursor” when usedin describing a protein refers to the primary product of translation ofmRNA; i.e., with pre- and propeptides still present. Pre- andpropeptides may be but are not limited to intracellular localizationsignals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“PCR” or “polymerase chain reaction” is well known by those skilled inthe art as a technique used for the amplification of specific DNAsegments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

The present invention concerns isolated polynucleotides comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding a polypeptide of at least 60 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:2, 4, 6, 43, 45, 47, 49, and 51; (b) a nucleotide sequenceencoding a polypeptide of at least 60 amino acids having at least 95%identity based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of SEQ ID NOs:9, 11, 13,15, 17, 19, 54 and 56; (c) a nucleotide sequence encoding a polypeptideof at least 60 amino acids having at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs:22, 24, 26, 28, and 59; (d) anucleotide sequence encoding a polypeptide of at least 60 amino acidshaving at least 95% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:31, 62, and 64; and (e) a nucleotide sequence encoding apolypeptide of at least 60 amino acids having at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:34, 36, 38, 40, 68, 70,and 72. It is preferred that the identity be at least 85%, it ispreferable if the identity is at least 90%, it is more preferred thatthe identity be at least 95%. This invention also relates to theisolated complement of such polynucleotides, wherein the complement andthe polynucleotide consist of the same number of nucleotides, and thenucleotide sequences of the complement and the polynucleotide have 100%complementarity.

Preferably, the isolated polynucleotide of the claimed inventioncomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs:1, 3, 5, 42, 44, 46, 48, 50, 8, 10, 12, 14, 16, 18, 53, 55,21, 23, 25, 27, 58, 30, 61, 63, 33, 35, 37, 39, 67, 69, and 71.

Nucleic acid fragments encoding at least a portion of several plantamino acid biosynthetic enzymes have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other aspartic semialdehyde dehydrogenases,diaminopimelate decarboxylases, homoserine kinases, cysteine γ synthasesor cystathionine β-lyases, either as cDNAs or genomic DNAs, could beisolated directly by using all or a portion of the instant nucleic acidfragments as DNA hybridization probes to screen libraries from anydesired plant employing methodology well known to those skilled in theart. Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis). Moreover, an entire sequence can be used directly tosynthesize DNA probes by methods known to the skilled artisan such asrandom primer DNA labeling, nick translation, end-labeling techniques,or RNA probes using available in vitro transcription systems. Inaddition, specific primers can be designed and used to amplify a part orall of the instant sequences. The resulting amplification products canbe labeled directly during amplification reactions or labeled afteramplification reactions, and used as probes to isolate full length cDNAor genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 30 (preferably at least 40, most preferably at least 60)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 42, 44, 46, 48, 50, 8, 10,12, 14, 16, 18, 59, 61, 21, 23, 25, 27, 64, 30, 33, 35, 37, 39, 53, 55,and 57 and the complement of such nucleotide sequences may be used insuch methods to obtain a nucleic acid fragment encoding a substantialportion of an amino acid sequence of a polypeptide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of an aspartic semialdehydedehydrogenase, diaminopimelate decarboxylase, homoserine kinase,cysteine synthase, or cystathionine β-lyase polypeptide, preferably asubstantial portion of a plant aspartic semialdehyde dehydrogenase,diaminopimelate decarboxylase, homoserine kinase, cysteine synthase, orcystathionine β-lyase polypeptide, comprising the steps of: synthesizingan oligonucleotide primer comprising a nucleotide sequence of at least30 (preferably at least 40, most preferably at least 60) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 42, 44, 46, 48, 50, 8, 10, 12, 14, 16,18, 53, 55, 21, 23, 25, 27, 58, 30, 61, 63, 33, 35, 37, 39, 67, 69, and71, and the complement of such nucleotide sequences; and amplifying anucleic acid fragment (preferably a cDNA inserted in a cloning vector)using the oligonucleotide primer. The amplified nucleic acid fragmentpreferably will encode a portion of an aspartic semialdehydedehydrogenase, diaminopimelate decarboxylase, homoserine kinase,cysteine synthase, or cystathionine β-lyase polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment, this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of free amino acids inthose cells. Specifically, the enzymes of the present invention formpart of the pathway towards the biosynthesis of lysine, threonine,methionine, cysteine and isoleucine. In particular, altering the leveland/or function of cystathionine beta-lyase will result in changes inthe rate of methionine biosynthesis. Altering the level and/or functionof diaminopimelate decarboxylase will result in changes in the rate oflysine biosynthesis. Altering the level and/or function ofaspartate-semialdehyde dehydrogenase will result in changes in thelysine, methionine, or threonine content, especially in wheat. Alteringthe level of cysteine γ synthase will result in changes in the rate ofcysteine and/or methionine biosynthesis; using this gene it will also bepossible to control sulfur metabolism. Altering the level of homoserinekinase may be used to regulate threonine and methionine levels.Polypeptides encoding at least a portion of aspartic semialdehydedehydrogenase, diaminopimelate decarboxylase, homoserine kinase,cysteine synthase, or cystathionine β-lyase may also be used inherbicide identification and design.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one which allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

In another embodiment, the present invention concerns anaspartic-semialdehyde dehydrogenase polypeptide of at least 50 aminoacids comprising at least 70% identity based on the Clustal method ofalignment compared to a polypeptide selected from the group consistingof SEQ ID NOs:2, 4, 6, 43, 45, 47, 49, and 51, a diaminopimelatedecarboxylase polypeptide of at least 60 amino acids comprising at least95% identity based on the Clustal method of alignment compared to apolypeptide selected from the group consisting of SEQ ID NOs:9, 11, 13,15, 17, 19, 60, and 62, a homoserine kinase polypeptide of at least 60amino acids comprising at least 70% identity based on the Clustal methodof alignment compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:22, 24, 26, 28, and 65, a cysteine synthasepolypeptide of at least 60 amino acids comprising at least 90% identitybased on the Clustal method of alignment compared to a polypeptide ofSEQ ID NO:31, or a cystathionine β-lyase polypeptide of at least 60amino acids comprising at least 85% identity based on the Clustal methodof alignment compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:34, 36, 38, 40, 54, 56, and 58.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded plant biosynthetic enzymes. An example of a vector for highlevel expression of the instant polypeptides in a bacterial host isprovided (Example 10).

Additionally, the instant polypeptides can be used as a target tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze various steps in a pathwayleading to production of several essential amino acids. Accordingly,inhibition of the activity of one or more of the enzymes describedherein could lead to inhibition of plant growth. Thus, the instantpolypeptides could be appropriate for new herbicide discovery anddesign.

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and used as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al.(1995) Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995)Plant Cell 7:75-84). The latter approach may be accomplished in twoways. First, short segments of the instant nucleic acid fragments may beused in polymerase chain reaction protocols in conjunction with amutation tag sequence primer on DNAs prepared from a population ofplants in which Mutator transposons or some other mutation-causing DNAelement has been introduced (see Bensen, supra). The amplification of aspecific DNA fragment with these primers indicates the insertion of themutation tag element in or near the plant gene encoding the instantpolypeptides. Alternatively, the instant nucleic acid fragment may beused as a hybridization probe against PCR amplification productsgenerated from the mutation population using the mutation tag sequenceprimer in conjunction with an arbitrary genomic site primer, such asthat for a restriction enzyme site-anchored synthetic adaptor. Witheither method, a plant containing a mutation in the endogenous geneencoding the instant polypeptides can be identified and obtained. Thismutant plant can then be used to determine or confirm the naturalfunction of the instant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries Isolation and Sequencing of cDNAClones

cDNA libraries representing mRNAs from various corn, rice, soybean, andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean, and Wheat LibraryTissue Clone cen1 Corn Endosperm 12 Days After Pollinationcen1.pk0061.d4 cen3n Corn Endosperm 20 Days After Pollination*cen3n.pk0067.a3 cpe1c Corn pooled BMS treated with chemicals related tocpe1c.pk009.b24 phosphatase** cr1n Corn Root From 7 Day Seedlings*cr1n.pk0009.g4 cr1n Corn Root From 7 Day Seedlings* cr1n.pk0103.d8 p0003Corn Premeiotic Ear Shoot, 0.2-4 cm p0003.cgpha22r:fis p0005 CornImmature Ear p0005.cbmei71r p0014 Corn Leaves 7 and 8 from PlantTransformed with p0014.ctuui39r G-protein Gene, C. heterostrophusResistant p0016 Corn Tassel Shoots (0.1-1.4 cm), Pooled p0016.ctscp83rp0075 Corn Shoot And Leaf Material From p0075.cslab16r Dark-Grown 7Day-Old Seedlings p0109 Corn Leaves From Les9 Transition Zone and Les9Mature p0109.cdadg47r Lesions, Pooled*** p0125 Corn Anther Prophase 1*p0125.czaay16r rca1c Rice Nipponbare Callus rca1c.pk005.k3 rl0n RiceLeaf 15 Days After Germination* rl0n.pk0013.b9 rlr12 Rice Leaf 15 DaysAfter Germination, 12 Hours After rlr12.pk0026.g1 Infection of StrainMagaporthe grisea 4360-R-62 (AVR2-YAMO) rlr48 Rice Leaf 15 Days AfterGermination 48 Hours After rlr48.pk0003.d12 Infection of StrainMagaporthe grisea 4360-R-62 (AVR2-YAMO) se3 Soybean Embryo 13 Days AfterFlowering sdp3c.pk001.o15 sdp3c Soybean Developing Pods 8-9 mm se3.05h06ses8w Mature Soybean Embryo 8 Weeks After Subculture ses8w.pk0020.b5ses9c Soybean Embryogenic Suspension ses9c.pk001.a15:fis sfl1 SoybeanImmature Flower sfl1.pk0012.c4 sfl1 Soybean Immature Flowersfl1.pk0122.f9 sr1 Soybean Root From 10 Day Old Seedlings sr1.pk0132.c1wdk1c Wheat Developing Kernel, 3 Days After Anthesis wdk1c.pk014.n5:fiswl1n Wheat Leaf from 7 Day Old Seedling* wl1n.pk0065.f2 wlk1 WheatSeedlings 1 hour After Fungicide Treatment**** wlk1.pk0012.c2 wr1 WheatRoot From 7 Day Old Seedlings wr1.pk0004.c11 wr1 Wheat Root From 7 DayOld Seedlings wr1.pk0091.g6 *These libraries were normalized essentiallyas described in U.S. Pat. No. 5,482,845. **Chemicals used includedokadaic acid, cyclosporin A, calyculin A, and cypermethrin, all of whichare commercially available from Molecular Biology supply sourcesincluding Calbiochem-Novabiochem Corp. ***Les9 mutants reviewed in “Anupdate on lesion mutants” Hoisington (1986) Maize Genetic Coop. NewsLett. 60: 50-51. ****Application of6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone; synthesis and methods ofusing this compound are described in USSN 08/545,827, incorporatedherein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prismdye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism Collections) and assembled usingPhred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrapis a public domain software program which re-reads the ABI sequencedata, re-calls the bases, assigns quality values, and writes the basecalls and quality values into editable output files. The Phrap sequenceassembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle).

Example 2 Identification of cDNA Clones

cDNA clones encoding plant amino acid biosynthetic enzymes wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

ESTs submitted for analysis are compared to the genbank database asdescribed above. ESTs that contain sequences more 5- or 3-prime can befound by using the BLASTn algorithm (Altschul et al (1997) Nucleic AcidsRes. 25:3389-3402.) against the DuPont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described in Example 1. Homologous genes belonging todifferent species can be found by comparing the amino acid sequence of aknown gene (from either a proprietary source or a public database)against an EST database using the tBLASTn algorithm. The tBLASTnalgorithm searches an amino acid query against a nucleotide databasethat is translated in all 6 reading frames. This search allows fordifferences in nucleotide codon usage between different species, and forcodon degeneracy.

Example 3 Characterization of cDNA Clones Encoding AspartateSemialdehyde Dehydrogenase

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs toaspartate semialdehyde dehydrogenase from Synechocystis sp. (DDJBAccession No. D64006; NCBI General Identifier No. 1001379) or Legionellapneumophila (GenBank Accession No. AF034213; NCBI General Identifier No.2645882). Shown in Table 3 are the BLAST results for individual ESTs(“EST”), or for the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toAspartate Semialdehyde Dehydrogenase BLAST pLog Score Synechocystis sp.Legionella pneumophila Clone Status GI 1001379 GI 2645882rlr48.pk0003.d12 FIS 51.00 36.00 wr1.pk0004.c11 EST 67.96 44.74sfl1.pk0122.f9 EST 6.60

The sequence of the entire cDNA insert in clone sfl1.pk0122.f9 wasdetermined, RACE PCR was used to obtain the 5′ portion of the riceaspartate semialdehyde dehydrogenase, and further sequencing andsearching of the DuPont proprietary database allowed the identificationof a corn and other a soybean, and wheat clones encoding aspartatesemialdehyde dehydrogenase. The BLASTX search using the EST sequencesfrom clones listed in Table 4 revealed similarity of the polypeptidesencoded by the cDNAs to aspartate semialdehyde dehydrogenase fromAquifex aeolicus (NCBI General Identifier No. 6225258). Shown in Table 4are the BLAST results for the sequences of contigs assembled from two ormore ESTs (“Contig”), or the sequences encoding the entire proteinderived from either the entire cDNA inserts comprising the indicatedcDNA clones or contigs assembled from 5′ RACE PCR and the sequence ofthe entire cDNA insert in the indicated cDNA clone (“CGS”):

TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous toAspartate Semialdehyde Dehydrogenase BLAST pLog Score Clone StatusAquifex aeolicus GI 6225258 Contig of: Contig 78.70 cpe1c.pk009.b24p0003.cgpha22r:fis p0016.ctscp83r p0075.cslab16r 5′ RACE PCR + CGS 89.20rlr48.pk0003.d12:fis ses9c.pk001.a15:fis CGS 87.40 sfl1.pk0122.f9:fisCGS 88.10 wdk1c.pk014.n5:fis CGS 91.50

FIG. 2 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:2, 4, 6, 43, 45, 47, 49, and 51 with the Legionellapneumophila sequence (NCBI General Identifier No. 2645882; SEQ ID NO:7)and the Aquifex aeolicus sequence (NCBI General Identifier No. 6225258;SEQ ID NO:52). The data in Table 5 presents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6,43, 45, 47, 49, and 51 with the Legionella pneumophila sequence (NCBIGeneral Identifier No. 2645882; SEQ ID NO:7) and the Aquifex aeolicussequence (NCBI General Identifier No. 6225258; SEQ ID NO:52).

TABLE 5 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toAspartate Semialdehyde Dehydrogenase amino acid Percent Identity toClone SEQ ID NO. 2645882 6225258 rlr48.pk0003.d12 2 42.1 45.6wr1.pk0004.c11 4 42.3 44.8 sfl1.pk0122.f9 6 29.1 25.6 Contig of: 43 41.245.9 cpe1c.pk009.b24 p0003.cgpha22r:fis p0016.ctscp83r p0075.cslab16r 5′RACE PCR + 45 43.2 47.0 rlr48.pk0003.d12:fis ses9c.pk001.a15:fis 47 43.549.1 sfl1.pk0122.f9:fis 49 41.2 45.6 wdk1c.pk014.n5:fis 51 43.2 49.4

As seen in FIG. 2, the amino acid sequence shown in SEQ ID NO:2 isidentical to amino acids 181 through 375 of SEQ ID NO:45; the sequenceshown in SEQ ID NO:4 is identical to amino acids 173 through 374 of thesequence shown in SEQ ID NO:51; the sequence shown in SEQ ID NO:6 isidentical to amino acids 1 through 86 of the sequence shown in SEQ IDNO:49; there are 5 amino acid differences between the sequences shown inSEQ ID NO:47 and SEQ ID NO:49; there are 18 amino acid differencesbetween amino acids 89 through 375 of the sequence shown in SEQ ID NO:43and the sequence shown in SEQ ID NO:45; and there are 15 differencesbetween the amino acid sequences shown in SEQ ID NO:45 and in SEQ IDNO:51.

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989)CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a corn aspartate semialdehydedehydrogenase, a substantial portion and an entire rice aspartatesemialdehyde dehydrogenase, a portion and an entire wheat aspartatesemialdehyde dehydrogenase, and a portion and an two entire soybeanaspartate semialdehyde dehydrogenases.

Example 4 Characterization of cDNA Clones Encoding DiaminopimelateDecarboxylase

The BLASTX search using the EST sequences from clones listed in Table 6revealed similarity of the polypeptides encoded by the cDNAs todiaminopimelate decarboxylase from Aquifex aeolicus (GenBank AccessionNo. AE000728 and NCBI General Identifier No. 2983642) and Pseudomonasaeruginosa (GenBank Accession No. M23174 and NCBI General Identifier No.118304). Shown in Table 6 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), or the sequences of FISs encoding anentire protein (“CGS”):

TABLE 6 BLAST Results for Sequences Encoding Polypeptides Homologous toDiaminopimelate Decarboxylase BLAST pLog Score GI 2983642 GI 118304Clone Status (A. aeolicus) (P. aeruginosa) cen3n.pk0067.a3 FIS 58.2256.00 cr1n.pk0103.d8 CGS 75.25 79.12 rl0n.pk0013.b9 FIS 46.40 44.00sr1.pk0132.c1 FIS 44.70 39.15 wlk1.pk0012.c2 EST 20.48 19.05

An additional soybean clone, sdp3c.pk001.o15, was identified as sharinghomology with sr1.pk0132.cl. BLASTX search using the nucleotidesequences from clone sdp3c.pk001.o15 revealed similarity of the proteinsencoded by the cDNA to diaminopimelate decarboxylase from Pseudomonasfluorescens (EMBO Accession No. Y12268; NCBI General Identifier No.1929095). This EST yields a pLog value of 8.66 versus the Pseudomonasfluorescens sequence.

The sequence of the entire cDNA insert in clones sdp3c.pk001.o15 andwlk1.pk0012.c2 was determined. The BLASTX search using the EST sequencesfrom clones listed in Table 7 revealed similarity of the polypeptidesencoded by the cDNAs to diaminopimelate decarboxylase from Aquifexaeolicus (NCBI General Identifier No. 6225241) or by the Arabidopsisthaliana contig containing similarity with diaminopimelatedecarboxylases (NCBI General Identifier No. 9279586). Shown in Table 7are the BLAST results for the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), or the sequences of FISsencoding the entire protein (“CGS”):

TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous toDiaminopimelate Decarboxylase BLAST Clone Status Homolog pLog Scoresdp3c.pk001.o15:fis CGS GI 6225241 (A. aeolicus) 76.40wlk1.pk0012.c2:fis FIS GI 9279586 (A. thaliana) 94.40

FIG. 3 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:9, 11, 13, 15, 17, 19, 54, and 56 with the Pseudomonasaeruginosa sequence (NCBI General Identifier No. 118304; SEQ ID NO:20)and the Arabidopsis thaliana sequence (NCBI General Identifier No.9279586, SEQ ID NO:57). The data in Table 8 presents a calculation ofthe percent identity of the amino acid sequences set forth in SEQ IDNOs:9, 11, 13, 15, 17, 19, 54, and 56 with the Pseudomonas aeruginosasequence (NCBI General Identifier No. 118304; SEQ ID NO:20) and theArabidopsis thaliana sequence (NCBI General Identifier No. 9279586; SEQID NO:57).

TABLE 8 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toDiaminopimelate Decarboxylase Amino acid Percent Identity to Clone SEQID NO. 118304 9279586 cen3n.pk0067.a3 9 34.0 82.2 cr1n.pk0103.d8 11 35.970.6 rl0n.pk0013.b9 13 32.4 76.8 sr1.pk0132.c1 15 29.7 86.1wlk1.pk0012.c2 17 42.5 93.2 sdp3c.pk001.o15 19 41.9 87.1sdp3c.pk001.o15:fis 54 32.5 74.9 wlk1.pk0012.c2:fis 56 32. 84.9

The amino acid sequence set forth in SEQ ID NO:19 is identical to aminoacids 112 through 173 of the amino acid sequence set forth in SEQ IDNO:54. The amino acid sequence set forth in SEQ ID NO:17 is identical toamino acids 24 through 96 of the amino acid sequence set forth in SEQ IDNO:56.

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989)CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of one corn, one rice, two soybean and onewheat diaminopimelate decarboxylases and entire corn and soybeandiaminopimelate decarboxylases.

Example 5 Characterization of cDNA Clones Encoding Homoserine Kinase

The BLASTX search using the EST sequences from clones listed in Table 9revealed similarity of the polypeptides encoded by the cDNAs tohomoserine kinase from Methanococcus jannaschii (GenBank Accession No.U67553 and NCBI General Identifier No. 1591748). Shown in Table 9 arethe BLAST results for individual ESTs (“EST”) or for the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones (“FIS”):

TABLE 9 BLAST Results for Sequences Encoding Polypeptides Homologous toHomoserine Kinase BLAST pLog Score Clone Status GI 1591748(Methanococcus jannaschii) cr1n.pk0009.g4 FIS 19.30 rca1c.pk005.k3 EST15.21 ses8w.pk0020.b5 FIS 35.30 wl1n.pk0065.f2 EST 5.68

The sequence of the entire cDNA insert in clone rca1c.pk005.k3 wasdetermined. The BLASTX search using the EST sequences from clones listedin Table 10 revealed similarity of the polypeptides encoded by the cDNAsto homoserine kinase from Arabidopsis thaliana (NCBI General IdentifierNo. 4927412). Shown in Table 10 are the BLAST results for the sequencesof the entire cDNA inserts comprising the indicated cDNA clone (“FIS”):

TABLE 10 BLAST Results for Sequences Encoding Polypeptides Homologous toHomoserine Kinase BLAST pLog Score Clone Status 4927412 (Arabidopsisthaliana) rca1c.pk005.k3:fis FIS 88.40

FIG. 4 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:22, 24, 26, 28, and 59 with the Methanococcus jannaschiisequence (NCBI General Identifier No. 1591748; SEQ ID NO:29) and theArabidopsis thaliana sequence (NCBI General Identifier No. 4927412; SEQID NO:60). The data in Table 11 presents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:22, 24, 26,28, and 59 with the Methanococcus jannaschii sequence (NCBI GeneralIdentifier No. 1591748; SEQ ID NO:29) and the Arabidopsis thalianasequence (NCBI General Identifier No. 4927412; SEQ ID NO:60).

TABLE 11 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toHomoserine Kinase SEQ ID Percent Identity to Clone NO. NCBI GI 1591748NCBI GI 4927412 cr1n.pk0009.g4 22 25.1 65.4 rca1c.pk005.k3 24 48.8 67.1ses8w.pk0020.b5 26 28.0 65.7 wl1n.pk0065.f2 28 29.8 67.9rca1c.pk005.k3:fis 59 28.6 65.9

The amino acid sequence set forth in SEQ ID NO:24 is identical to aminoacids 18 through 99 of the amino acid sequence set forth in SEQ IDNO:59.

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989)CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a corn and a wheat homoserine kinase, aportion and an entire rice homoserine kinase, and an entire soybeanhomoserine kinase.

Example 6 Characterization of cDNA Clones Encoding Cysteine Synthase

The BLASTX search using the EST sequences from the clone listed in Table12 revealed similarity of the polypeptides encoded by the cDNAs tocysteine synthase from Citrullus lanatus (DDJB Accession No. D28777,NCBI General Identifier No. 540497). Shown in Table 12 are the BLASTresults for the sequences of the entire cDNA inserts comprising theindicated cDNA clones encoding the entire protein (“CGS”):

TABLE 12 BLAST Results for Sequences Encoding Polypeptides Homologous toCysteine γ Synthase BLAST pLog Score Clone Status NCBI GI 540497(Citrullus lanatus) se3.05h06 CGS 182.64

Further sequencing and searching of the DuPont proprietary databaseallowed the identification of corn and rice clones encoding polypeptideswith similarities to cysteine γ synthase. The BLAST search using thesequences from clones listed in Table 13 revealed similarity of thepolypeptides encoded by the cDNAs to cysteine γ synthase from Spinaciaoleracea (NCBI General Identifier No. 416869) and Solanum tuberosum(NCBI General Identifier No. 11131628). Shown in Table 13 are the BLASTresults for the sequences of the entire cDNA inserts comprising theindicated cDNA clones encoding the entire protein (“CGS”):

TABLE 13 BLAST Results for Sequences Encoding Polypeptides Homologous toCysteine γ Synthase BLAST pLog Score NCBI GI 416869 NCBI GI 11131628Clone Status (Spinacia oleracea) (Solanum tuberosum) Contig of: CGS158.00 157.00 cco1n.pk083.j4 chp2.pk0016.b1 cpd1c.pk004.b20cr1n.pk0083.c5 csi1.pk0003.g6 p0126.cnlcb49r rls6.pk0068.b7:fis CGS161.00 163.00

FIG. 5 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:31, 62, and 64 with the Citrullus lanatus sequence (NCBIGeneral Identifier No. 540497; SEQ ID NO:32), Spinacia oleracea (NCBIGeneral Identifier No. 416869; SEQ ID NO:65), and the Solanum tuberosumsequence (NCBI General Identifier No. 11131628; SEQ ID NO:66). The datain Table 14 presents a calculation of the percent identity of the aminoacid sequences set forth in SEQ ID NOs:31, 62, and 64 with the Citrulluslanatus sequence (NCBI General Identifier No. 540497; SEQ ID NO:32),Spinacia oleracea (NCBI General Identifier No. 416869; SEQ ID NO:65),and the Solanum tuberosum sequence (NCBI General Identifier No.11131628; SEQ ID NO:66).

TABLE 14 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toCysteine γ Synthase Percent Identity to Amino acid NCBI NCBI NCBI CloneSEQ ID NO. GI 540497 GI 416869 GI 11131628 se3.05h06 31 87.1 72.3 76.9Contig of: 62 73.8 71.3 69.7 cco1n.pk083.j4 chp2.pk0016.b1cpd1c.pk004.b20 cr1n.pk0083.c5 csi1.pk0003.g6 p0126.cnlcb49rrls6.pk0068.b7:fis 64 73.2 72.6 72.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode entire corn, rice, and soybean cysteine γ synthases. Thesesequences represent the first corn, rice, and soybean sequences encodingcysteine γ synthase known to Applicant.

Example 7 Characterization of cDNA Clones Encoding Cystathione β-Lyase

The BLASTX search using the EST sequences from clones listed in Table 15revealed similarity of the polypeptides encoded by the cDNAs tocystathionine β-lyase from Arabidopsis thaliana (GenBank Accession No.L40511; NCBI General Identifier No. 1708993). Shown in Table 15 are theBLAST results for individual ESTs (“EST”), the sequences of the entirecDNA inserts comprising the indicated cDNA clones (“FIS”), or thesequences of FISs encoding the entire protein (“CGS”):

TABLE 15 BLAST Results for Sequences Encoding Polypeptides Homologous toCystathione β-Lyase BLAST pLog Score Clone Status 1708993 (A. thaliana)cen1.pk0061.d4 FIS 50.41 rlr12.pk0026.g1 EST 39.00 sfl1.pk0012.c4 CGS33.85 wr1.pk0091.g6 EST 52.52

The sequence of the entire cDNA insert in the clone wr1.pk0091.g6 wasdetermined, RACE PCR was used to obtain the 5′ portion of the ricecystathionine β-lyase, and further sequencing and searching of theDuPont proprietary database allowed the identification of other corn andwheat clones encoding cystathionine β-lyase. The BLASTX search using theEST sequences from clones listed in Table 16 revealed similarity of thepolypeptides encoded by the cDNAs to cystathionine β-lyase fromArabidopsis thaliana (GenBank Accession No. L40511; NCBI GeneralIdentifier No. 1708993). Shown in Table 16 are the BLAST results for thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), or the sequences encoding the entire protein derivedfrom contigs assembled from the sequences of more than two ESTs, thesequence of contigs assembled from the entire cDNA inserts comprisingthe indicated cDNA clones and 5′ RACE PCR or an EST (“Contig*”):

TABLE 16 BLAST Results for Sequences Encoding Polypeptides Homologous toCystathione β-Lyase BLAST pLog Score Clone Status 1708993 Contig of:Contig* >180.00 cen1.pk0061.d4 p0005.cbmei71r p0014.ctuui39rp0109.cdadg47r p0125.czaay16r 5′ RACE PCR + Contig* 178.00rlr12.pk0026.g1:fis wr1.pk0091.g6:fis FIS 177.00

FIG. 6 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:34, 36, 38, 40, 68, 70, and 72 with the Arabidopsis thalianasequence (NCBI General Identifier No. 1708993; SEQ ID NO:41). The datain Table 17 presents a calculation of the percent identity of the aminoacid sequences set forth in SEQ ID NOs:34, 36, 38, 40, 68, 70, and 72with the Arabidopsis thaliana sequence (NCBI General Identifier No.1708993; SEQ ID NO:41).

TABLE 17 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toCystathione β-Lyase Percent Identity to Clone SEQ ID NO. 1708993(Arabidopsis thaliana) cen1.pk0061.d4 34 83.0 rlr12.pk0026.g1 36 76.0sfl1.pk0012.c4 38 72.2 wr1.pk0091.g6 40 71.8 Contig of: 68 66.8cen1.pk0061.d4 p0005.cbmei71r p0014.ctuui39r p0109.cdadg47rp0125.czaay16r 5′ RACE PCR + 70 66.2 rlr12.pk0026.g1:fiswr1.pk0091.g6:fis 72 66.2

The amino acid sequence set forth in SEQ ID NO:34 is identical to aminoacids 248 through 470 of the amino acid sequence set forth in SEQ IDNO:68. The amino acid sequence set forth in SEQ ID NO:36 is identical toamino acids 152 through 226 of the amino acid sequence set forth in SEQID NO:70. The amino acid sequence set forth in SEQ ID NO:40 is identicalto amino acids 3 through 133 of the amino acid sequence set forth in SEQID NO:72.

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode an entire soybean cystathionine β-lyase, a substantial portionand an entire corn and rice cystathionine β-lyases, a portion and asubstantial portion of a wheat cystathionine β-lyase.

Example 8 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or Sma I) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes Nco I and Sma I and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNco I-Sma I fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb Sal I-Nco I promoter fragment of the maize 27 kD zeingene and a 0.96 kb Sma I-Sal I fragment from the 3′ end of the maize 10kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNAcan be ligated at 15° C. overnight, essentially as described (Maniatis).The ligated DNA may then be used to transform E. coli XL1-Blue(Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can bescreened by restriction enzyme digestion of plasmid DNA and limitednucleotide sequence analysis using the dideoxy chain termination method(Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmidconstruct would comprise a chimeric gene encoding, in the 5′ to 3′direction, the maize 27 kD zein promoter, a cDNA fragment encoding theinstant polypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35 S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 9 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 10 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% low melting agarose gel. Buffer and agarose contain 10μg/ml ethidium bromide for visualization of the DNA fragment. Thefragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 11 Evaluating Compounds for Their Ability to Inhibit theActivity of Plant Biosynthetic Enzymes

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 10, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptides may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instant polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. Examples of assays for many of these enzymes can be found inMethods in Enzymology Vol. V, (Colowick and Kaplan eds.) Academic Press,New York or Methods in Enzymology Vol. XVII, (Tabor and Tabor eds.)Academic Press, New York. Specific examples may be found in thefollowing references, each of which is incorporated herein by reference:aspartic semialdehyde dehydrogenase may be assayed as described in Blacket al. (1955) J. Biol. Chem. 213:39-50, or Cremer et al. (1988) J. Gen.Microbiol. 134:3221-3229; diaminopimelate decarboxylase may be assayedas described in Work (1962) in Methods in Enzymology Vol. V, (Colowickand Kaplan eds.) 864-870, Academic Press, New York or Cremer et al.(1988) J. Gen. Microbiol. 134:3221-3229; homoserine kinase may beassayed as described in Aarnes (1976) Plant Sci. Lett. 7:187-194;cysteine synthase may be assayed as described in Thompson et al. (1968)Biochem. Biophys. Res. Commun. 31: 281-286 or Bertagnolli et al. (1977)Plant Physiol. 60:115-121; and cystathionine β-lyase may be assayed asdescribed in Giovanelli et al. (1971) Biochim. Biophys. Acta 227:654-670or Droux et al. (1995) Arch. Biochem Biophys. 316:585-595.

1. An isolated polynucleotide that encodes a plant cysteine γ synthasehaving amino acid sequence identity of at least 95% based on the Clustalmethod of alignment when compared to a polypeptide selected from thegroup consisting of SEQ ID NOs:31, 62, and
 64. 2. The polynucleotide ofclaim 1 wherein the polynucleotide encodes a polypeptide selected fromthe group consisting of SEQ ID NOs: NOs:31, 62, and
 64. 3. Thepolynucleotide of claim 1, wherein the polynucleotide comprises anucleotide sequence selected from the group consisting of SEQ ID NO:30,61, and
 63. 4. An isolated complement of the polynucleotide of claim 1,wherein (a) the complement and the polynucleotide consist of the samenumber of nucleotides, and (b) the nucleotide sequences of thecomplement and the polynucleotide have 100% complementarity.
 5. Anisolated nucleic acid molecule that (1) comprises at least 180nucleotides (2) remains hybridized with a polynucleotide having anucleotide sequence selected from the group consisting of SEQ ID NO:30,61, and 63 under a wash condition of 0.1×SSC, 0.1% SDS, and 65° C., andencodes a plant cysteine γ synthase.
 6. A cell comprising thepolynucleotide of claim
 1. 7. The cell of claim 6, wherein the cell isselected from the group consisting of a yeast cell, a bacterial cell anda plant cell.
 8. A transgenic plant comprising the polynucleotide ofclaim
 1. 9. A method for transforming a cell comprising introducing intoa cell the polynucleotide of claim
 1. 10. A method for producing atransgenic plant comprising (a) transforming a plant cell with thepolynucleotide of claim 1, and (b) regenerating a plant from thetransformed plant cell.
 11. A method for producing a polynucleotidefragment comprising (a) selecting a nucleotide sequence comprised by thepolynucleotide of claim 1, and (b) synthesizing a polynucleotidefragment containing the nucleotide sequence.
 12. The method of claim 11,wherein the fragment is produced in vivo.
 13. A chimeric gene comprisingthe polynucleotide of claim 1 operably linked to at least one regulatorysequence.
 14. A method for altering the level of cysteine γ synthaseexpression in a host cell, the method comprising: (a) Transforming ahost cell with the chimeric gene of claim 13; and (b) growing thetransformed cell from step (a) under conditions suitable for theexpression of the chimeric gene.
 15. A method for evaluating a compoundfor its ability to inhibit the activity of a plant cysteine γ synthase,the method comprising the steps of: (a) transforming a host cell with achimeric gene comprising a polynucleotide of claim 1, operably linked toat least one regulatory sequence; (b) growing the transformed host cellunder conditions that are suitable for expression of the chimeric genewherein expression of the chimeric gene results in production of theplant biosynthetic enzyme encoded by the operably linked nucleic acidfragment in the transformed host cell; (c) optionally purifying theplant biosynthetic enzyme polypeptide expressed by the transformed hostcell; (d) treating the plant biosynthetic enzyme with a compound to betested; (e) comparing the activity of the plant biosynthetic enzyme thathas been treated with a test compound to the activity of an untreatedplant biosynthetic enzyme polypeptide; and (f) selecting the compoundthat inhibits the activity of cysteine γ synthase.
 16. An isolatedpolynucleotide comprising: (a) a nucleotide sequence encoding apolypeptide having plant amino acid biosynthetic activity, wherein thepolypeptide has an amino acid sequence identity of at least 90% based onthe Clustal method of alignment, when compared to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 9, 11, 13, 15, 17, 19,22, 24, 26, 28, 34, 36, 38, and 40, or (b) a complement of thenucleotide sequence, wherein the complement and the nucleotide sequenceconsist of the same number of nucleotides and are 100% complementary.17. The isolated polynucleotide of claim 1, wherein the amino acidsequence of the polypeptide has at least 95% sequence identity based onthe Clustal method of alignment when compared to a polypeptide selectedfrom the group consisting of SEQ ID NOs: 2, 4, 6, 9, 11, 13, 15, 17, 19,22, 24, 26, 28, 34, 36, 38, and
 40. 18. The isolated polynucleotide ofclaim 1, wherein the polynucleotide encodes a polypeptide selected fromthe group consisting of SEQ ID NOs: 2, 4, 6, 9, 11, 13, 15, 17, 19, 22,24, 26, 28, 34, 36, 38, and
 40. 19. The isolated polynucleotide of claim1, wherein the polynucleotide comprises a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 8, 10, 12, 14, 16, 18,21, 23, 25, 27, 33, 35, 37, and
 39. 20. An isolated nucleic acidmolecule that (1) comprises at least 180 nucleotides and (2) remainshybridized with a polynucleotide having a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 8, 10, 12, 14, 16, 18,21, 23, 25, 27, 33, 35, 37, and 39 under a wash condition of 0.1×SSC,0.1% SDS, and 65° C.
 21. A cell comprising the polynucleotide of claim16.
 22. The cell of claim 21, wherein the cell is selected from thegroup consisting of a yeast cell, a bacterial cell, and a plant cell.23. A transgenic plant comprising the polynucleotide of claim
 16. 24. Amethod for transforming a cell comprising introducing into a cell thepolynucleotide of claim
 16. 25. A method for producing a transgenicplant comprising: (a) transforming a plant cell with the polynucleotideof claim 16, and (b) regenerating a plant from the transformed plantcell.
 26. A method for producing a polynucleotide fragment comprising:(a) selecting a nucleotide sequence comprised by the polynucleotide ofclaim 16, and (b) synthesizing a polynucleotide fragment containing thenucleotide sequence.
 27. The method of claim 26, wherein the fragment isproduced in vivo.
 28. A chimeric gene comprising the polynucleotide ofclaim 16 operably linked to at least one regulatory sequence.
 29. Amethod for altering the level of expression of a plant amino acidbiosynthetic enzyme in a host cell, the method comprising: (a)transforming a host cell with the chimeric gene of claim 28; and (b)growing the transformed cell from step (a) under conditions suitable forthe expression of the chimeric gene.